Application Of New Fusion Sequence In Long-term GLP-1 Drugs Development

As we all know,the peptide-like drugs are easily hydrolyzed by protease and have generally short half-life in the body,increasing the administration frequency and dosage to get therapeutic effects.Indeed,it not only causes great pain to patients,but also loads severe economic burden.Therefore,more and more R & D institutions explore novel long-term strategies to extend the half-life of peptide drugs,such as the substitution of special amino acids,PEG modification,esterification,and long-acting protein sequence fusion.The present research have demonstrated the serum albumin binding(SABA),XTEN and PAS sequences can significantly enhance the half-life of peptide drugs,but there have not been related reports in China.In this study,the expression of the fusion proteins was investigated by combining the three long-acting sequences with GLP-1 to evaluate pharmacodynamics,which served as a reference for the development of long-acting peptide drug in China.In this paper,three long-acting sequences,SABA,XTEN and PAS sequence,were applied to design the long-acting GLP-1 fusion proteins,including 18 long-acting SABA-GLP-1s with different linkers and combinations,four long-acting XTEN-GLP-1s with different lengths and combinations,and two long-acting PAS-GLP-1s with different lengths and combinations.The expressions of these 24 long-acting GLP-1 fusion sequences were identified in Escherichia coli.Through optimizing induction conditions in fermentation,the expression level of fusion protein was increased,and the methods of separation and purification were utilized according to different fusion proteins.In addition,the detection method was established to verify the bioactivities of Albiglutide and GLP-1(7-37)at the cellular level,meanwhile all the long-acting GLP-1 fusion proteins suffered from activity assay and comparative analysis.The results indicated that SABA-GLP-1 fusion proteins were expressed in the form of inclusion body.Although denaturation and renaturation were implemented at the later stage,the purification procedure of renatured SABA-GLP-1s was relatively simple.Besides,SABA-GLP-1s had significant differences in expression level and bioactive value due to diverse linkers.Their biological activities were generally not high in vitro,in which the highest biological activity of fusion protein expressed in S12 strain reached 9.296 nmol/L.Both XTEN-GLP-1 and PAS-GLP-1 fusion proteins existed in the form of soluble protein.Despite the complicated purification process,their biological activities were generally higher than those of SABA-GLP-1s in vitro and were 5.068 nmol/L and 2.820 nmol/L,respectively.This study substantiated three long-acting fusion proteins were obtained by recombinant expression in Escherichia coli.Obviously,peptide drug was combined with the three long-acting sequences,SABA,XTEN and PAS sequence,to maintain its bioactivity to some extent.Moreover,the changes of long-acting sequence length,linker and combination significantly affect the biological activities of fusion proteins.Thus,their biological activities can be further improved by optimizing sequence,linker and combination.